Efficient production of oligosaccharides using metabolically engineered microorganisms

ABSTRACT

The present invention relates to the enzymatic synthesis of oligosaccharides, particularly, sialylated oligosaccharides comprising the carbohydrate moieties of the gangliosides GM3, GD3, and G.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 14/608,944, filed Jan. 29, 2015, now U.S. Pat. No. 9,688,954, whichis a Continuation application of U.S. application Ser. No. 12/723,264,filed Mar. 12, 2010, now U.S. Pat. No. 8,975,054, which is a Divisionalapplication of U.S. patent application Ser. No. 11/447,287, filed Jun.6, 2006, now U.S. Pat. No. 7,820,422, which claims the benefit of U.S.Provisional Application No. 60/690,837, filed Jun. 16, 2005. Thecontents of these applications are herein incorporated by reference intheir entirety.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format in priority U.S. application Ser. No.11/447,287 on Sep. 27, 2006 and is hereby incorporated by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to the enzymatic synthesis ofoligosaccharides, particularly, sialylated oligosaccharides comprisingthe carbohydrate moieties of the gangliosides GM3, GD3, and GT3.

BACKGROUND OF THE INVENTION

It is now well-established that oligosaccharides play an importantbiological role especially as regards to the activity and function ofproteins; thus, they serve to modulate the half-life of proteins, andoccasionally they are involved in the structure of the protein.Oligosaccharides play an essential role in antigen variability (forexample blood groups), and in certain bacterial infections such as thosecaused by Neisseria meningitides.

As oligosaccharides are usually obtained in a low yield by purificationstarting from natural sources, the synthesis of oligosaccharides hasbecome a major challenge of carbohydrate chemistry. In particular, it isa goal to supply sufficient amounts of well-characterizedoligosaccharides, required for fundamental research or for any otherpotential applications.

The synthesis of complex oligosaccharides of biological interest may beperformed chemically, enzymatically or microbiologically. Despite thedevelopment of new chemical methods for synthesizing oligosaccharides inthe course of the last 20 years, the chemical synthesis ofoligosaccharides remains very difficult on account of the numerousselective protection and deprotection steps, the lability of theglycoside linkages, the difficulties in obtaining regiospecificcouplings, and the low production yields.

GD3 (Neu5Acα-8Neu5Acα-3Galβ-4GlcCer) is a minor ganglioside found inmost normal tissues in higher vertebrates including humans. The GD3level has been shown to increase during some pathological situations,such as cancers (glioma, melanoma) and to have an important role intumor angiogenesis Zeng, et al. Cancer Res, 60:6670 (2000). Anti-GD3monoclonal antibodies have been shown to inhibit the growth of humanmelanoma cells both in vitro and in vivo (Birkle, et al. Biochimie, 85,455 (2003); Ruf, et al. Int J Cancer, 108, 725 (2004)). In normal cells,GD3 is a cell death effector, activating the mitochondrial-dependentapoptosome in response to apoptotic stimuli (Fernandez-Checa, BiochemBiophys Res Commun, 304, 471 (2003)). In addition, GD3 has aproapoptotic function by suppressing the nuclear factor-Kb-dependentsurvival pathway (Colell, et al. Faseb J, 15, 1068 (2001)).

Chemical synthesis of the oligosaccharide moiety of gangliosides isdifficult to achieve (Castro-Palomino et al., Chemistry, 7, 2178 (2001))but new efficient biotechnological techniques have recently beendeveloped for the synthesis of GM3, GM2 and GM1 oligosaccharides (Priemet al. Glycobiology, 12, 235 (2002) and Antoine et al., Chembiochem, 4,406 (2003)). The GM3 oligosaccharide (Neu5Acα-3Galβ-4Glc) wassynthesized by a metabolically engineered Escherichia coli strain whichoverexpressed the Neisseria meningitidis genes for α-3 sialyltransferaseand CMP-Neu5Ac synthase. Lactose and neuraminic acid (Neu5Ac) weresupplied as exogenous precursors and actively internalized by E. coli'sβ-galactosidase and Neu5Ac permease. To prevent catabolism of theprecursors, a mutant strain devoid of both β-galactosidase and Neu5Acaldolase activities was used.

Despite advances in the art, new biosynthetic methods for producingdesired oligosaccharides are needed. The present invention addressesthese and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of producing oligosaccharides byfermentative growth of microorganisms. In particular, the inventionrelates to a method of synthesis of the oligosaccharide moieties ofgangliosides selected from:

GM3 (Neu5Acα-3Galβ-4Glc),

GD3 (Neu5Acα-8Neu5Acα-3Galβ-4Glc), and

GT3 (Neu5Acα-8Neu5Acα-8Neu5Acα-3Galβ-4Glc)

using bifunctional Cainpylobacter jejuni CstII sialyltransferases.

This method may be extended to the production of GM3, GD3 and GT3 byreacting the above oligosaccharide moieties with ceramide.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the metabolically engineered pathway of II³(Neu5Ac)₂lac(GD3) biosynthesis in Escherichia coli K12. Lactose and Neu5Ac, whichwere internalized by the specific permeases LacY and NanT, could not bedegraded because of β-galactosidase (LacZ) and aldolase (NanA)inactivation. Neu5Ac was converted into a nucleotide activated form(CMP-Neu5Ac) by CMP-Neu5Ac synthase and then transferred onto lactose bythe α-3 sialyltransferase activity of CstII to form sialyllactose. Asecond Neu5Ac was transferred onto the first sialic acid by the α-8sialyltransferase activity of CstII to form II³(Neu5Ac)₂lac. CTP,cytidine triphosphate; PPi, inorganic pyrophosphate.

FIG. 2 shows a production of oligosaccharides in a high-cell densityculture of strain TA 15. The arrow indicates the start of induction andthe addition of lactose (3 mM) and Neu5Ac (6 mM): (Δ) cell growth; (X)lactose, (⋄) Neu5Ac; (●) II³(Neu5Ac)-lac (1); (▪) II³(Neu5Ac)₂-lac (2);(♦) II³(Neu5Ac)₃-lac (3); (◯) sum of II³(Neu5Ac)-lac, II³(Neu5Ac)₂-lacand II³(Neu5Ac)₃-lac. Concentrations are given in mM.

FIG. 3 shows the structures of GM3 (II³(Neu5Ac)-lac, 1); GD3(II³(Neu5Ac)₂-lac, 2) GT3 (II³(Neu5Ac)₃-lac, 3).

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention provides cell-based methods for enzymaticallysynthesizing sialylated oligosaccharides. Also provided are reactionmixtures, expression cassettes, and recombinant cells that are useful inmethods for synthesizing sialylated oligosaccharides. Typically, thecells are grown in a medium that includes the precursors lactose andNeu5Ac. Production of the sialylated oligosaccharides occurs duringfermentative growth of the cells, as the precursors are taken up by thecells and metabolized to form the desired oligosaccharides, i.e., GM3,GD3 or GT3 oligosaccharides.

The methods of the invention rely on the general approach disclosed inWO 01/04341, an English translation of which is NZ516808. In general,the sialylated product saccharides are produced by growing amicroorganism that comprises an enzymatic system for synthesizing anactivated sialic acid (e.g., a CMP-sialic acid synthase polypeptide) anda bifunctional C. jejuni CstII sialyltransferase in the presence of aprecursor of sialic acid and lactose, under conditions such that anactivated sialic acid molecule is synthesized and transfer of the sialicacid moiety from the activated sialic acid molecule is catalyzed by thesialyltransferase to produce the sialylated product saccharide. Alsoprovided by the invention are recombinant cells that can be used in themethods, as well as reaction mixtures that include the recombinant cellsand are useful for producing the product sugars.

The nomenclature and general laboratory procedures required to practicethe present invention are well known to those of skill in the art Theseprocedures can be found, for example, in Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989.

Definitions

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid (often abbreviated asNeu5Ac, NeuAc, or NANA). A second member of the family isN-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetylgroup of NeuAc is hydroxylated. A third sialic acid family member is2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J. Biol.Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265: 21811-21819(1990)). Also included are 9-substituted sialic acids such as a9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

The term “bifunctional Campylobacter jejuni CstII sialyltransferase”refers to a sialyltransferase which exhibits both α2-3 and αa2-8sialyltransferase activities. In some embodiments, the CstIIsialyltransferase from ATCC Accession No. 43438 is used.

An “acceptor substrate” or an “acceptor saccharide” for aglycosyltransferase is an oligosaccharide moiety that can act as anacceptor for a particular glycosyltransferase. When the acceptorsubstrate is contacted with the corresponding glycosyltransferase andsugar donor substrate, and other necessary reaction mixture components,and the reaction mixture is incubated for a sufficient period of time,the glycosyltransferase transfers sugar residues from the sugar donorsubstrate to the acceptor substrate. For example, an acceptor substratefor the sialyltransferases used in the methods of the invention islactose Gaβ1,4-Glc.

A “donor substrate” for glycosyltransferases is an activated nucleotidesugar. Such activated sugars generally consist of uridine, guanosine,and cytidine monophosphate derivatives of the sugars (UMP, GMP and CMP,respectively) or diphosphate derivatives of the sugars (UDP, GDP andCDP, respectively) in which the nucleoside monophosphate or diphosphateserves as a leaving group. For example, a donor substrate forsialyltransferases used in the methods of the invention is CMP-Neu5Ac.

A “culture medium” refers to any liquid, semi-solid or solid media thatcan be used to support the growth of a microorganism used in the methodsof the invention. In some embodiments, the microorganism is a bacteria,e.g., E. coli. Media for growing microorganisms are well known, see,e.g., Sambrook et al. and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1998Supplement) (Ausubel). Media can be rich media, e.g., Luria broth orterrific broth, or synthetic or semi-synthetic medium, e.g., M9 medium.In some preferred embodiments the growth medium comprises lactose andsialic acid.

“Commercial scale” refers to gram scale production of a sialylatedproduct saccharide in a single reaction. In preferred embodiments,commercial scale refers to production of greater than about 50, 75, 80,90 or 100, 125, 150, 175, or 200 grams.

The term “operably linked” refers to functional linkage between anucleic acid expression control sequence (such as a promoter, signalsequence, or array of transcription factor binding sites) and a secondnucleic acid sequence, wherein the expression control sequence affectstranscription and/or translation of the nucleic acid corresponding tothe second sequence.

A “heterologous polynucleotide” or a “heterologous gene”, as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Thus, a heterologous sialyltransferase gene in a cell includes agene that is endogenous to the particular host cell but has beenmodified. Modification of the heterologous sequence may occur, e.g., bytreating the DNA with a restriction enzyme to generate a DNA fragmentthat is capable of being operably linked to a promoter. Techniques suchas site-directed mutagenesis are also useful for modifying aheterologous sequence.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of affecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally, transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used. Transcriptiontermination signals, enhancers, and other nucleic acid sequences thatinfluence gene expression, can also be included in an expressioncassette. When more than one heterologous protein is expressed in amicroorganism, the genes encoding the proteins can be expressed on asingle expression cassette or on multiple expression cassettes that arecompatible and can be maintained in the same cell. As used herein,expression cassette also encompasses nucleic acid constructs that areinserted into the chromosome of the host microorganism. Those of skillare aware that insertion of a nucleic acid into a chromosome can occur,e.g., by homologous recombination. An expression cassette can beconstructed for production of more than one protein. The proteins can beregulated by a single promoter sequence, as for example, an operon. Ormultiple proteins can be encoded by nucleic acids with individualpromoters and ribosome binding sites.

The term “isolated” refers to material that is substantially oressentially free from components which interfere with the activitybiological molecule. For cells, saccharides, nucleic acids, andpolypeptides of the invention, the term “isolated” refers to materialthat is substantially or essentially free from components which normallyaccompany the material as found in its native state. Typically, isolatedsaccharides, oligosaccharides, proteins or nucleic acids of theinvention are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% pure as measured by band intensity on a silver stained gelor other method for determining purity. Purity or homogeneity can beindicated by a number of means well known in the art, such aspolyacrylarnide gel electrophoresis of a protein or nucleic acid sample,followed by visualization upon staining. For certain purposes highresolution will be needed and HPLC or a similar means for purificationutilized. For oligosaccharides, e.g., sialylated products, purity can bedetermined using, e.g., thin layer chromatography, HPLC, or massspectroscopy.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80% or 85%, most preferably at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide or aminoacid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In a mostpreferred embodiment, the sequences are substantially identical over theentire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent substitutions” or “silentvariations,” which are one species of “conservatively modifiedvariations.” Every polynucleotide sequence described herein whichencodes a polypeptide also describes every possible silent variation,except where otherwise noted. Thus, silent substitutions are an impliedfeature of every nucleic acid sequence which encodes an amino acid. Oneof skill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques. In someembodiments, the nucleotide sequences that encode the enzymes arepreferably optimized for expression in a particular host cell (e.g.,yeast, mammalian, plant, fungal, and the like) used to produce theenzymes.

Similarly, “conservative amino acid substitutions,” in one or a fewamino acids in an amino acid sequence are substituted with differentamino acids with highly similar properties are also readily identifiedas being highly similar to a particular amino acid sequence, or to aparticular nucleic acid sequence which encodes an amino acid. Suchconservatively substituted variations of any particular sequence are afeature of the present invention. Individual substitutions, deletions oradditions which alter, add or delete a single amino acid or a smallpercentage of amino acids (typically less than 5%, more typically lessthan 1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.See, e.g., Creighton (1984) Proteins, W.H. Freeman and Company.

Bifunctional Sialyltransferases and Accessory Enzymes

As noted above, bifunctional sialyltransferases are used in the methodsof the invention. Nucleic acids encoding such enzymes have been isolatedfrom C. jejuni and are disclosed in U.S. Pat. Nos. 6,699,705 and6,503,744 and WO/02074942. Exemplary C. jejuni strains which can be usedas sources of bifunctional sialyltransferases include OH4384 (nucleicacid sequences are found in GenBank accessions AR271700 and AX934425),OH4382, O:10 (nucleic acid sequences are found in GenBank accessionsAR271701 (SEQ ID NO 2), O:23, and O:41 (nucleic acid sequences are foundin GenBank accessions AR271702 (SEQ ID NO 3) and AX934429 (SEQ ID NO4)). It shall be understood that conservatively modified variations asdefined above SEQ ID NO 1, 2, 3 and 4 may be applied herein.

Glycosyltransferase reactions require a nucleotide sugar which serves assugar donor. Enzymes that are involved in synthesis of a nucleotidesugar or synthesis of the sugar are also called accessory enzymes.Accessory enzymes include those enzymes that are involved in theformation of a nucleotide sugar.

The sialyltransferases used in the methods of the invention require aCMP-sialic acid molecule, i.e., an activated sialic acid molecule, toserve as a donor of a sialic acid moiety. In some embodiments, therecombinant cells of the invention can naturally produce the CMP-sialicacid molecule that serves as a sugar donor for the sialyltransferaseproduced by the cell, as well as the nucleotide to which the sialic acidmoiety is attached. However, some cells do not naturally producesufficient amounts of either or both of the CMP or the sialic acid toproduce the desired quantities of product saccharide In such situations,the recombinant cells of the invention can contain at least oneheterologous gene that encodes an accessory enzyme, involved insynthesis of CMP-sialic acid.

Thus in some embodiment of the invention, a gene that encodes aCMP-sialic acid synthetase (EC 2.7.7.43, CMP-N-acetylneuraminic acidsynthetase) is introduced into the cell. Sources for such genes are wellknown to those of skill in the art. In some preferred embodiments, thegene for CMP-sialic acid synthase from Neisseria meningitis MC58 is used(see e.g., Gilbert et al. (1996) J. Biol. Chem., 271:28271-28276 and WO01/04341).

Host Cells

The recombinant cells of the invention are generally made by creating orotherwise obtaining a polynucleotide that encodes the particularenzyme(s) of interest, placing the polynucleotide in an expressioncassette under the control of a promoter and other appropriate controlsignals, and introducing the expression cassette into a cell. More thanone of the enzymes can be expressed in the same host cells using avariety of methods. For example, a single extrachromosomal vector caninclude multiple expression cassettes or more that one compatibleextrachromosomal vector can be used maintain an expression cassette in ahost cell. Expression cassettes can also be inserted into a host cellchromosome, using methods known to those of skill in the art. Those ofskill will recognize that combinations of expression cassettes inextrachromosomal vectors and expression cassettes inserted into a hostcell chromosome can also be used. Other modification of the host cell,described in detail below, can be performed to enhance production of thedesired oligosaccharide. For example, the microorganism may be LacY+ andNanT+ allowing active transport of lactose and sialylic acid.

The recombinant cells of the invention are generally microorganisms,such as, for example, yeast cells, bacterial cells, or fungal cells.Examples of suitable cells include, for example, Azotobacter sp. (e.g.,A. vinelandii), Pseudomonas sp., Rhizobium sp., Erwinia sp., Bacillussp., Streptomyces sp., Escherichia sp. (e.g., E. coli), and Klebsiellasp., among many others. The cells can be of any of several genera,including Saccharomyces (e.g., S. cerevisiae), Candida (e.g., C. utilis,C. parapsilosis, C. krusei, C. versatilis. C. lipolytica, C.zeylanoides, C. guilhermondii, C. albicans, and C. humicola), (e.g., P.farinosa and P. ohmeri), Torulopsis (e.g., T. candida, T. sphaerica, T.xylinus, T. famata, and T. versatilis), Debaryomyces (e.g., D.subglobosus, D. cantarellii, D. globosus, D. hansenii, and D.japonicus), Zygosaccharomyces (e.g., Z. rouxii and Z. bailii),Kluyveromyces (e.g., K. marxianus), Hansenula (e.g., H. anomala and H.jadinii), and Brettanomyces (e.g., B. lambicus and B. anomalus).

Promoters for use in E. coli include the T7, trp, or lambda promoters. Aribosome binding site and preferably a transcription termination signalare also provided. For expression of heterologous proteins inprokaryotic cells other than E. coli, a promoter that functions in theparticular prokaryotic species is required. Such promoters can beobtained from genes that have been cloned from the species, orheterologous promoters can be used. For example, the hybrid trp-lacpromoter functions in Bacillus in addition to E. coli. Methods oftransforming prokaryotes other than E. coli are well known. For example,methods of transforming Bacillus species and promoters that can be usedto express proteins are taught in U.S. Pat. Nos. 6,255,076 and6,770,475.

In yeast, convenient promoters include GAL1-10 (Johnson and Davies(1984) Mol. Cell. Biol. 4:1440-1448) ADH2 (Russell et al. (1983) J.Biol. Chem. 258:2674-2682), PHO5 (EMBO J. (1982) 6:675-680), and MFa(Herskowitz and Oshima (1982) in The Molecular Biology of the YeastSaccharomyces (eds. Strathern, Jones, and Broach) Cold Spring HarborLab., Cold Spring Harbor, N.Y., pp. 181-209). Another suitable promoterfor use in yeast is the ADH2/GAPDH hybrid promoter as described inCousens et al., Gene 61:265-275 (1987). For filamentous fungi such as,for example, strains of the fungi Aspergillus (McKnight et al., U.S.Pat. No. 4,935,349), examples of useful promoters include those derivedfrom Aspergillus nidulans glycolytic genes, such as the ADH3 promoter(McKnight et al., EMBO J, 4: 2093 2099 (1985)) and the tpiA promoter. Anexample of a suitable terminator is the ADH3 terminator (McKnight etal.).

In some embodiments, the polynucleotides are placed under the control ofan inducible promoter, which is a promoter that directs expression of agene where the level of expression is alterable by environmental ordevelopmental factors such as, for example, temperature, pH, anaerobicor aerobic conditions, light, transcription factors and chemicals. Suchpromoters are referred to herein as “inducible” promoters, which allowone to control the timing of expression of the glycosyltransferase orenzyme involved in nucleotide sugar synthesis. For E. coli and otherbacterial host cells, inducible promoters are known to those of skill inthe art. These include, for example, the lac promoter. A particularlypreferred inducible promoter for expression in prokaryotes is a dualpromoter that includes a tac promoter component linked to a promotercomponent obtained from a gene or genes that encode enzymes involved ingalactose metabolism (e.g., a promoter from a UDPgalactose 4-epimerasegene (galE)).

Inducible promoters for other organisms are also well known to those ofskill in the art. These include, for example, the arabinose promoter,the lacZ promoter, the metallothionein promoter, and the heat shockpromoter, as well as many others.

The construction of polynucleotide constructs generally requires the useof vectors able to replicate in bacteria. A plethora of kits arecommercially available for the purification of plasmids from bacteria.For their proper use, follow the manufacturer's instructions (see, forexample, EasyPrepJ, FlexiPrepJ, both from Pharmacia Biotech;StrataCleanJ, from Stratagene; and, Q1Aexpress Expression System,Qiagen). The isolated and purified plasmids can then be furthermanipulated to produce other plasmids, and used to transfect cells.Cloning in Streptomyces or Bacillus is also possible.

Selectable markers are often incorporated into the expression vectorsused to construct the cells of the invention. These genes can encode agene product, such as a protein, necessary for the survival or growth oftransformed host cells grown in a selective culture medium. Host cellsnot transformed with the vector containing the selection gene will notsurvive in the culture medium. Typical selection genes encode proteinsthat confer resistance to antibiotics or other toxins, such asampicillin, neomycin, kanamycin, chloramphenicol, or tetracycline.Alternatively, selectable markers may encode proteins that complementauxotrophic deficiencies or supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.Often, the vector will have one selectable marker that is functional in,e.g., E. coli, or other cells in which the vector is replicated prior tobeing introduced into the target cell. A number of selectable markersare known to those of skill in the art and are described for instance inSambrook et al., supra. A preferred selectable marker for use inbacterial cells is a kanamycin resistance marker (Vieira and Messing,Gene 19: 259 (1982)). Use of kanamycin selection is advantageous over,for example, ampicillin selection because ampicillin is quickly degradedby β-lactamase in culture medium, thus removing selective pressure andallowing the culture to become overgrown with cells that do not containthe vector.

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques as described inthe references cited above, Isolated plasmids or DNA fragments arecleaved, tailored, and re-ligated in the form desired to generate theplasmids required. To confirm correct sequences in plasmids constructed,the plasmids can be analyzed by standard techniques such as byrestriction endonuclease digestion, and/or sequencing according to knownmethods. Molecular cloning techniques to achieve these ends are known inthe art. A wide variety of cloning and in vitro amplification methodssuitable for the construction of recombinant nucleic acids arewell-known to persons of skill.

A variety of common vectors suitable for constructing the recombinantcells of the invention are well known in the art. For cloning inbacteria, common vectors include pBR322 derived vectors such aspBLUESCRIPT™, and λ-phage derived vectors. In yeast, vectors includeYeast Integrating plasmids (e.g., Ylp5) and Yeast Replicating plasmids(the YRp series plasmids) and pGPD-2.

The methods for introducing the expression vectors into a chosen hostcell are not particularly critical, and such methods are known to thoseof skill in the art. For example, the expression vectors can beintroduced into prokaryotic cells, including E. coli, by calciumchloride transformation, and into eukaryotic cells by calcium phosphatetreatment or electroporation. Other transformation methods are alsosuitable.

In some embodiments, production of oligosaccharides is enhanced bymanipulation of the host microorganism. For example, in E. coli, breakdown of sialic acid can be minimized by using a host strain that hasdiminished CMP-sialic acid synthase activity (NanA-). In E. coli,CMP-sialic acid synthase appears to be a catabolic enzyme. Diminishingthe sialic acid degradative pathway in a host cell can be accomplishedby disrupting the N-acetylneuraminate lyase gene (NanA, Accession numberAE000402 region 70-963). Introduction of a sialyltransferase gene intothese mutant strains results in a recombinant cell that is capable ofproducing large amounts of a sialylated product saccharide.

In some embodiments, the microorganisms are manipulated to enhancetransport of an acceptor saccharide into the cell. For example, wherelactose is the acceptor saccharide, E. coli cells that express oroverexpress the LacY permease can be used. Also in E. coli, when lactoseis the acceptor saccharide or an intermediate in synthesizing thesialylated product, lactose breakdown can be minimized by using hostcells that are LacZ-.

Methods for Producing Product Saccharides

The invention also provides methods in which the host cells are used toprepare product sialylated oligosaccharides. The culture medium thenincludes lactose, sialic acid and possibly other precursors to donorsubstrates or acceptor substrates.

Those of skill will recognize that culture medium for microorganisms canbe e.g., rich mediums, such as Luria broth, animal free Luria broth, orTerrific broth or synthetic medium or semi-synthetic medium, such as M9medium.

Other components of the growth medium include the acceptor saccharide,lactose and sialic acid. Concentrations of the acceptor saccharide canbe between 1 and 100 mM. In a preferred embodiment, the acceptorsaccharide concentration is about 3 mM. Sialic acid can be present at aconcentration between 1 and 100 mM, usually about 6 mM. In one preferredembodiment, Neu5Ac is present at or continuously supplied to maintain aconcentration between 2 mM and 60 mM, such as about 6 mM, in the cellculture medium. Also, lactose is present at or continuously supplied tomaintain a concentration between 1 mM and 30 mM, such as about 3 mM.

The methods of the invention can be used for producing sialylatedoligosaccharides that are labeled with or enriched in radioisotopes;such oligosaccharides are extremely useful for fundamental biological orconformational analysis studies. The invention thus relates to a methodfor producing an oligosaccharide that is labeled with at least oneradioisotope. In these embodiments, the culture medium includessubstrates labeled said radioisotope and/or in the presence of a saidprecursor labeled with said radioisotope. The radioisotopes arepreferably chosen from the group composed of 14C, 13C, 3H, 358, 32p,33p.

The methods of the invention can also be used to activatedoligosaccharides that may be used for the chemical synthesis ofglycoconjugates or glycopolymers. The lactose acceptor can thus bemodified such that glucose residue is replaced with an allyl group, saidprecursor now being allyl-13-D galactoside rather than lactose. Forexample, the double bond of the allyl group is chemically modified byaddition, oxidation or ozonolysis reactions.

Methods and culture media for growth of microorganisms are well known tothose of skill in the art. Culture can be conducted in, for example,aerated spinner or shaking culture, or, more preferably, in a fermentor.

The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the product.Standard, well known techniques for recovery of glycosylated saccharidessuch as thin or thick layer chromatography, column chromatography, ionexchange chromatography, or membrane filtration can be used. It ispreferred to use membrane filtration, more preferably utilizing areverse osmotic membrane, or one or more column chromatographictechniques for the recovery as is discussed hereinafter and in theliterature cited herein.

Therapeutic and Other Uses

Sialylated oligosaccharides made according to the invention are usefulin a wide range of therapeutic and diagnostic applications. They may beused, for example, as an agent for blocking cell surface receptors inthe treatment of a host of diseases. As noted above, theoligosaccharides used for the chemical synthesis of glycoconjugates(e.g., glycolipids) or glycopolymers. The oligosaccharides or theglycoconjugates may be used, for example, as nutritional supplements,antibacterial agents, anti-metastatic agents and anti-inflammatoryagents. The invention thus relates to an oligosaccharide according tothe invention as a medicinal product in which the sialylatedoligosaccharide or glycoconjugate is used to prepare a pharmaceuticalcomposition. Methods for preparing pharmaceutical compositions are wellknown in the art.

For example, immunoadsorbents made with the trisaccharideNeu5Acα-8Neu5Acα-3Gal have recently been shown to deplete anti-GQ1 bantibodies in autoimmune neuropathy sera from patients suffering fromthe Miller Fisher syndrome (Willison et al. Brain, 127, 680 (2004)). Thelarge scale preparation of the GD3 oligosaccharide using the presentinvention is thus useful in the preparation of pharmaceuticalcomposition in immunoadsorption therapies for the treatment of thissyndrome. Therefore, the invention also contemplates a method fortreating patients suffering from the Miller Fisher syndrome comprisingdepleting anti-GQ1b antibodies in sera from said patients usingimmunoadsorption with the large scale preparation of the oligosaccharideobtained according the method as defined above.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.Citations are incorporated herein by reference.

EXAMPLES

The metabolically engineered pathway for the biosynthesis of the GD3oligosaccharide is described in FIG. 1 (see also Antoine et al. Anger.Chem Int. Ed. 44:2-4 (2005)). The host strain, JM107nanA⁻, was anEscherichia coli K12 JM107 strain derivative in which the Neu5Acaldolase activity was abolished by inactivating the nanA gene (Antoineet al. Chembiochem, 4, 406 (2003)). The cstII gene was amplified by PCRusing the genomic DNA of C. jejuni (ATCC 43438) as template and clonedinto a pUC18 plasmid to yield pUC-cstII. The II³(Neu5Ac)₂lac productionstrain TA15 was constructed by transforming the host strain JM107nanA″with two compatible plasmids: pUC18-cstII and pBBnsy (Priem et al.Glycobiology, 12, 235 (2002)) which was pBBRT-MCSI derivative carryingthe N. meningitidis gene for CMP-Neu5Ac synthase. The strain TA15 wascultured to high cell density with glycerol as the carbon source aspreviously described (Priem et al., supra). Lactose (3 mM) and Neu5Ac (6mM) were added at the beginning of the fed-batch phase, as well as IPTG,which was the inducer of the Plac promoter of the two plasmids. Theoligosaccharide content in the intracellular fraction of sampleswithdrawn at different cultivation times was analysed by HPAEC-PADanalysis (Supporting information Figure SI). Lactose and Neu5Actransiently accumulated in the intracellular fraction and were entirelyconsumed after 7 and 24 hours of incubation respectively (FIG. 2).Concurrently two compounds 1 and 2, putatively II³(Neu5Ac)lac andII³(Neu5Ac)₂1ac, were produced, reaching their maximal concentrations 7and 9 hours after induction, respectively. Concentrations of compounds 1and 2 then decreased during the final stage of culture and this decreasecorrelated with the appearance of compound 3 which had a longerretention time in HPAEC (supporting information Figure S1) and which wassupposedly believed to be II³(Neu5Ac)₃-lac. The structure of compounds1, 2, 3 was confirmed by NMR and mass spectrometry of purified products.The ¹³C assignments of 1 and 2 (supporting information table S1,spectrum A) were in close agreement with previously described data(Gilbert, et al. J Biol Chem, 275, 3896 (2000)). ¹³C assignment of 3 wasmade by crosschecking ¹³C NMR spectrum of compound 2 and of α-8 linkedtri-Neu5Ac F. Michon, et al. Biochemistry, 26, 8399 (1987)) (supportinginformation table S1, spectra B and C). In the positive mode, massspectroscopic analysis showed the presence of quasi molecular ions[M+Na]⁺ at m/z 656 and [(M+Na—H)+Na]⁺ at m/z 678 in the spectrum ofcompound 1 and quasi molecular ions [M+Na]⁺ at m/z 969 and[(M+Na—H)+Na]⁺ at m/z 991 in the spectrum of compound 2. The massspectrum of compound 3 showed one peak at m/z 1304 in the positive modecorresponding to the quasi molecular ion [(M+3Na—3H)+Na]⁺ and one peakat m/z 1258 in the negative mode corresponding to the quasi molecularion [(M+2Na—2H)−H]. The structures of the carbohydrate moieties of thegangliosides GM3 (II³(-Neu5Ac)-lac) (1), GD3 (II³(Neu5Ac)₂-lac) (2) andGT3 (II³(Neu5Ac)₃-lac) (3) are presented in FIG. 3.

The formation of compound 3 can be explained by a side activity of theCstII sialyltransferase which would be able to add a third Neu5Ac ontothe Neu5Ac of II³(Neu5Ac)₂-lac. The production of compound 3 wassomewhat unexpected because no such trisialylated structure has beendescribed in the lipooligosaccharides of C. jejuni and there is noreport of Cst-II being able to use a Neu5Acα-8Neu5Acα-3 motif as anacceptor. However in other species, such as humans,polysialogangliosides are synthesized by a single enzyme, GD3/GT3synthase (Nakayama et al. J Biol Chem, 271, 3684 (1996)). GT3 is theprecursor of C series gangliosides which are the major constituents inadult fish brain and are found abundantly in fetal brains of highervertebrates (Letinic, et al. Neuroscience, 86, 1 (1998)). They are alsofound in various neuroectodermal tumors and there is thus potentiallygreat interest in having easy access to the GT3 oligosaccharide.

By varying the lactose and NeuAc initial concentrations on one hand, andthe culture time on the other, it is possible to favor either theproduction of GD3 or GT3 oligosaccharides. In our culture conditions themaximal production yields of GD3 (0.83 g·L⁻¹) and GT3 (0.91 g·L⁻¹)oligosaccharides were observed 9 hours and 24 hours after inductionrespectively. The yields of purified products were much lower due to themany purification steps. The purification procedure has thus to beconsiderably improved if these compounds were to be used in applicationswhich require large quantities of material.

The cstII gene was amplified by PCR from C. jejuni ATCC43438 genome withthe Pfu Turbo DNA polymerase from Stratagene. A ribosome binding site(RBS) was inserted upstream of the original start codon in the forwardprimer. The PCR product was cloned into pCR4Blunt-TOPO (Invitrogen) andsubcloned into the EcoRI-PstI sites of pBluescript II KS (stratagene).

Sugars were quantified by high-performance anion-exchange chromatography(HPAEC) with a decade detector equipped with a carbopac PA10 column(DIONEX). Purified compounds 1, 2 and 3 were used as standards. Theelution program consisted of a linear gradient of sodium acetate from 0to 0.5 M in 45 minutes with an isocratic background of sodium hydroxide(100 mM). The flow rate was 0.8 mL/h.

Compounds 1 and 2 were purified from one liter of a strain TA15 cultureharvested 9 hours after induction. The intracellular oligosaccharideswere extracted from the cells and adsorbed on activated charcoal aspreviously described (Samain et al. Carbohydr Res, 302, 35(1997)). Theywere then separated by size exclusion chromatography on a Biogel P6column (4×100 cm) with NaNO₃ (50 mM) as the eluent and a flow rate of 45ml h⁻¹ (supporting information Figure S2). After desalting on a TSKHW40F/50F column (50×2, 1 cm) with water as the mobile phase and a flowrate of 4 ml min¹, the yields of pure compounds 1 and 2 were 49 mg and98 mg respectively.

Compound 3 was purified from one liter of a strain TA15 cultureharvested 24 hours after induction. The procedure was the same as forcompounds 1 and 2 except that complete purification required anadditional High Performance Liquid Chromatography step on an ionexchange SP250/10 nucleosil column (10×1 cm). NaNO₃ (150 mM) was used asthe eluent and the flow rate was 4 ml min⁻¹. After a last desalting stepon a TSK HW40F/50F column, the final yield of pure compound 3 was 15 mg.

ESI mass spectra were recorded on a ZQ Waters micromass spectrometer(capillary 3.5 kV, cone voltage 80V). The 1D NMR spectra ofII³(Neu5Ac)lac and II³(Neu5Ac)₂lac were recorded on a Bruker AVANCE 300spectrometer as previously described (Antoine et al. Chembiochem, 4, 406(2003)).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications (including GenBankaccessions), patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

What is claimed is:
 1. An Escherichia coli microorganism comprising aheterologous gene encoding a bifunctional Campylobacter jejunisialytransferase polypeptide that catalyzes the transfer of a sialylmoiety from an activated sialic acid molecule to lactose to formNeu5Acα-3Galβ-4Glc and a heterologous gene encoding a cytidinemonophospho-(CMP)-sialic acid synthetase; wherein the bifunctionalCampylobacter jejuni sialyltransferase polypeptide has at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NOS: 1, 2, 3, and
 4. 2. The microorganism of claim 1, wherein theheterologous gene encoding the CMP-sialic acid synthetase is fromNeisseria meningitidis.
 3. The microorganism of claim 1, wherein thebifunctional Campylobacter jejuni sialytransferase polypeptide is fromCampylobacter jejuni isolate deposited under American Type CultureCollection (ATCC) Accession No.
 43438. 4. The microorganism of claim 1,wherein the microorganism lacks Neu5Ac aldolase activity.
 5. Themicroorganism of claim 1, wherein the microorganism lacks βgalactosidase activity.
 6. The microorganism of claim 1, wherein themicroorganism is a LacY+ and NanT+ Escherichia coli allowing activetransport of lactose and sialylic acid.
 7. A cell culture mediumcomprising lactose and Neu5Ac and the microorganism of claim
 1. 8. Thecell culture medium of claim 7, wherein the Neu5Ac is present at orcontinuously supplied to maintain a concentration between 2 mM and 60mM.
 9. The cell culture medium of claim 8, wherein the Neu5Ac is presentat or continuously supplied to maintain a concentration of about 6 mM.10. The cell culture medium of claim 7, wherein the lactose is presentat or continuously supplied to maintain a concentration between 1 mM and30 mM.
 11. The cell culture medium of claim 10, wherein the lactose ispresent at or continuously supplied to maintain a concentration of about3 mM.
 12. A method of producing an oligosaccharide comprisingNeu5Acα-3Galβ-4Glc, the method comprising: (a) obtaining themicroorganism of claim 1; and (b) culturing the microorganism in aculture medium comprising lactose and Neu5Ac.
 13. The method of claim12, wherein the Neu5Ac is present at or continuously supplied tomaintain a concentration between 2 mM and 60 mM.
 14. The method of claim13, wherein the Neu5Ac is present at or continuously supplied tomaintain a concentration of about 6 mM.
 15. The method of claim 12,wherein and the lactose is present at or continuously supplied tomaintain a concentration between 1 mM and 30 mM.
 16. The method of claim15, wherein the lactose is present at or continuously supplied tomaintain a concentration of about 3 mM.
 17. A method of producing anoligosaccharide comprising Neu5Acα-3Galβ-4Glc according to claim 12,which also comprise production of Neu5Acα-8Neu5Acα-3Galβ-4Glc andNeu5Acα-8Neu5Acα-8Neu5Acα-3Galβ-4Glc.
 18. The microorganism of claim 1,wherein the heterologous gene encoding the CMP-sialic acid synthetase isfrom Neisseria meningitidis 406Y, Neisseria meningitidis M982B,Neisseria meningitidis MC58 or Neisseria gonorrhoeae F62.